During this reporting period July, August, and September, 2016), Dr. McNellis continued to work with USDA APHIS to obtain permitting to transfer a set of ‘Duncan’ grapefruit plants expressing the FLT-antiNodT fusion protein from Penn State University to Fort Detrick in Frederick, Maryland. These plants are to be tested for resistance to HLB using a psyllid-vectored inoculation system in a secure greenhouse. We anticipate approval during the next reporting period. In addition, Dr. McNellis’ team at Penn State continued to evaluate the solubility and stability of the FLT-antiNodT fusion protein in citrus extracts and presence of the FLT-antiNodT fusion protein in various plant tissues by protein gel immunoblotting. The FLT-antiNodT fusion protein appears to be produced and present in all tissues examined to date, although these tests are ongoing and will continue into the next reporting period. Dr. McNellis presented a poster describing the results of the project to date at the annual conference of the American Phytopathological Society in Tampa, FL, July 30 – August 4, 2016. A poster viewer at the conference had some suggestions as to how to determine whether the FLT-antiNodT fusion protein indeed binds to its target in vivo, and Dr. McNellis has developed an experimental plan for doing this, which will be initiated during the next reporting period.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. 1. To test for he unlikely but increasing possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. As of June 2016, trees were growing normally. Samples of nectar, honey, pollen, albedo, flavedo and flowers collected in spring time were were analyzed. Albedo, flavedo and flower buds all tested HLB+, whereas pollen, seeds, nectar and honey were all HLB-. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees. The trees have been placed in a greenhouse and continue currently under observation. PCR analyses were conducted in May 2016 and one of the 5 originally healthy trees tested positive, although clear visible symptoms were not evident. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. Although we continue to have issues with the background, we have established a standard curve and a correlation relationship between starch levels, PCR values, and polarized light readings. 4. Trees have been grafted for a substantial amount of time and some started showing HLB symptoms. However, given that analysis of this objective destroys the trees, more time is needed to be certain that HLB has taken root. PCR analyses was performed in May 2016 and no tree tested positive despite the now clear symptoms of HLB. In general, we continue to monitor all experiments realizing that CLas titer drops during the summer. All trees will be tested again in September and proper action taken depending on the results.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. 1. To test for he unlikely, but increasing, possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. As of June 2016, trees were growing normally. Trees tested in September 11, one tree testing HLB+, though a high PCR value. Samples of nectar, honey, pollen, albedo, flavedo and flowers collected in spring time had been analyzed previously. Albedo, flavedo and flower buds all tested HLB+, whereas pollen, seeds, nectar and honey were all HLB-. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees (5 pairs). The trees were placed in a greenhouse and kept under observation. PCR analyses were conducted once more in September 2016. At this time, 3 out of the 5 pairs of the initially healthy trees tested positive, although clear visible symptoms were not evident in all cases. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. Although we continue to have issues with the background, we have established a standard curve and a correlation relationship between starch levels, PCR values, and polarized light readings. 4. Trees have been grafted for a substantial amount of time and some are showing HLB symptoms. However, given that analysis of this objective destroys the trees, only trees with clear symptoms are tested. PCR analyses was conducted in one tree using using all leaves in 2 complete orthostichies. In this particular tree, there was no correlation between orthostichy and titer, although there PCR values were significantly lower in the laves above the infection point.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
During the reporting time period, we were in the process of hiring a scientist to work on project. Meanwhile, we have made following progresses for the proposed objectives: We analyzed and identified the gene sequences required for the projects and designed all proposed gene constructs. Most of these gene cassettes are currently under construction, with some being finished soon. These constructs will be used in citrus transformation to test their effects in promoting mature citrus transformation. We have also been evaluating the effects of a hormone related gene in improving micro-grafting efficiency using tobacco as a quick testing model plant. Our results demonstrate that the expression of that particular hormone related gene in rootstock plants can improve scion grafting success rate, reduce rootstock’s lateral bud release, and enhance rootstock’s root initiation. We will soon ship this gene to Dr. Janice Zale of the Mature Citrus Facility at the University of Florida for them to test its effects on citrus.
Citrus trees transformed with a chimera AMP and a thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Propagated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Twelve months after graft inoculation, Las titer was examined and compared in old leaves (most with HLB symptom), young expanded leaves (with or without HLB symptom) and fibrous roots of transgenic and control plants. Our results showed again that transgenic citrus expressing Mthionin has lower Las titer (200-1800X lower) compared to control and transgenic plant expressing chimera. These data suggest transgenic plants expressing thionin are promising for HLB resistance ( published in Frontiers in Plant Biology). Antibody against thionin has been produced for investigating the correlation of thionin expression and HLB resistance. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. Furthermore, the third generation chimeral peptides were designed based on citrus thionins, the vector construction were finished and citrus transformation are underway. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA was isolated and Las titer was tested. Our preliminary results showed that transgenic trees expressing NbFLS2 can reduced Las titer. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las tilter will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
Additional UF trees have been planted in the last quarter. Data collection continues across numerous experiments. A number of publications from UF and USDA have included data from these plantings. A test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for six years and new trees are being added every few months. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015. The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al, Plant Disease, 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree (Miles et al., 2016). All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has underway using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall led assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
The overall objective of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using Liberibacter crescens strain BT-1. We have accomplished that goal and used it to screen potential phage, tailocins and microbial compounds for activity against the model bacterium L. crescens BT-1. Since Liberibacter is a member of the Rhizobiaceae we have recently taken the approach that phylogenetically related microorganisms can share common surface components, such as phage receptor sites. Analysis of major outer proteins of Candidatus Liberibacter spp. , that are known to be expressed in planta and in the vector, identified several proteins that could potentially act as adsorption sites for phage and/or tailocins. Bioinformatics and structural analyses indicated that there is ~40% identity and ~61% homology of OmpA and TolC outer membrane proteins between Rhizobium spp., Agrobacterium spp. and Liberibacter spp. Therefore, our strategies has been to search for naturally occurring phages active against Rhizobium spp. and/or Agrobacterium spp. that may also show activity against Liberibacter spp. We have isolated a bank of three Agrobacterium spp. and 15 Rhizobium spp. phages. The Agrobacterium phages exhibit differential activity among Agrobacterium spp. hosts, as do the 15 Rhizobium phages, which indicates a diversity of receptor sites. One particular Rhizobium phage, R2phi3LR, forms plaques on both Agrobacterium spp. and Rhizobium spp. hosts, which is indicative of common receptor(s). As expected, the efficiency of plating for phage R2phi3LR was reduced 1000X when Rhizobium propagated phage was titered on Agrobacterium and vice versa. All purified phages are being increased to conduct plant studies that will evaluate the potential activity of the phages against Candidatus Liberibacter spp. .
This project is a continuation of CRDF 447 to evaluate effects of Metalized Reflective Mulch (MRM) and insecticides to protect newly planted trees from the Asian Citrus Psyllid (ACP). While the previous project demonstrated that trees planted over the MRM treatment exhibited less ACP populations, less HLB symptoms and greater growth, it did not include crop yield and fruit quality differences which is the focus of this project. Specifically the objectives of this project are: 1) To determine if grapefruit trees planted into beds covered with Metalized Reflective Mulch (MRM) come into viable crop production at a younger age than in conventional plantings based on yield differences. 2) To continue to document tree growth differences between the three treatments: bare ground (convention grower standard), compost applications, and MRM and to monitor (weekly) insect pest populations to determine the insect control benefits of the MRM. 3) To determine the HLB incidence with PCR analysis for each treatment to verify the effectiveness of MRM to bring young trees into production without HLB symptoms. 4) To determine tree condition by improvements of tree health attributable to MRM will be made by visual assessments and Normalized Difference Vegetation Index (NDVI) determined by aerial (UAV) imagery of the canopy to determine the tree condition for each treatment. 5) To record and document production costs and economic returns for each treatment. To accomplish these goals we have continued to make growth measurements for the following parameters: tree caliper, tree height, canopy diameter and canopy volume. In all these four growth measurement the MRM treatments were significantly greater than both the bare ground and compost treatments and in almost all case the increase of the MRM treatment was double that attributed to the compost treatment when bare ground was the control treatment. For example the increase in canopy volume for MRM was 137% greater than bare ground while compost was 72% greater. The Volumetric Water Content (VWC) for the three treatments was sampled monthly and the MRM consistently remained the highest for the three treatments. Tree health was evaluated visually as tree condition and the MRM treatment trees has the lowest percentage of weak trees (1.6%), Compost (3.8%) and Bare Ground (18.8%). Similar trends were observed for symptomatic conditions attributable to HLB. The MRM treatment continued to offer lower ACP counts for all life stages (eggs, nymphs and adults) based on weekly scouting. Two other insect pest populations were also observed to be lower in the MRM treatment were Diaprepes Root Weevil and Orange Dog larvae. A detailed spreadsheet has been created to track all the caretaking expenses for the trial block including all spray foliar spray applications, herbicide treatments, fertilizer applications, soil drenches as well as the associated materials. These caretaking events are entered as they are accomplished and are combined to yield the total production cost for each treatment. Pertaining to yield component of the trial we have met with Dr. Alan Wright and Mr. Jerry Britt (IRREC) to coordinate the upcoming harvest event to determine the average fruit yield and fruit size for each treatment by means of a portable scale and fruit sizer. The average fruit drop per tree was assessed for each treatment and yielded the following data: Bare Ground 3.6, Compost (UPD) 3.2 and MRM 2.3. Currently the overall tree condition trial is deemed to be excellent with very minor damage associated with Hurricane Matthew.
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of October 12, 2016, a total of 9,309 plants have passed through inoculation process. A total of 293,895 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. As reported in the last progress report and reiterated here, research recently showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of Ca. Liberibacter asiaticus by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant). Research results will soon be available in which we are comparing success rates using ACP colonies on lemon versus citron, and using ACP colonies from greenhouses versus walk-in chambers.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. T One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We have confirmed and analyzed the genome modified plants including off-targets. No side effect was observe. Publications from this project 1. Jia H, Zhang Y, Orbovic V, Xu J, White F, Jones J, Wang N. (2016) Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker. Plant Biotechnol J. doi: 10.1111/pbi.12677. 2. Jia, H., Orbovic, V., Jones, J.B. and Wang, N. 2016 Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating Xcc.pthA4:dCsLOB1.3 infection. Plant Biotechnol. J., 14: 1291 1301. doi: 10.1111/pbi.12495. 3. Jia H, Wang N. 2014 Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 9(4):e93806. doi: 10.1371/journal.pone.0093806
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We have confirmed and analyzed the genome modified plants including off-targets. No side effect was observe. The data has been summarized into one manuscript and submitted. We are currently focusing on generating EBE mutated plants in both alleles and generating plants which do not contain cas9 and sgRNA in the plant chromosome.
Core Citrus Transformation Facility (CCTF) moved to temporary location in the last week of July. The staff tried to minimize the impact of moving on the operation of the lab. We ended up canceling only half week worth of experiments. However, the location of the lab itself presented a challenge and has negatively affected productivity. Within the last two months, the death rate of micro-grafted positive transgenic shoots more than doubled. Some of previously grafted and established plants also exhibited signs of poor growth and few of them died. I have instructed the staff to do grafting in lab in different building and keep grafted shoots out of CCTF. We also consulted with the member of Mature Tissue Transformation who does grafting and got some advice from him. The orders continued to come to CCTF in high numbers. Within this quarter we have received 12 more orders. Altogether, we received 50 orders since the beginning of the year. Resistance to canker and HLB continue to dominate the orders placed at CCTF. CCTF produced 60 plants within the last quarter. Out of those plants, five were Carrizo citranges, three Swingle citrumelos, two Pineapple sweet orange, and 50 Duncan grapefruits. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. Those plants are very tall now and will require additional care soon. If they are supposed to be propagated by cuttings, that should be done very soon, since this type of propagation is not very successful when done during the months of November, December, and January.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter,cuttings have been initiated for selected transgenic lines. Molecular analyses has been completed and a wide variation in transgene response is being observed. Due to yet unexplained reasons, transgenic lines expressing the ASAL transgene are performing better horticulturally than lines with expressing the APA transgene. Rooted cuttings are expected to be available in the fall.
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